ES2555858T3 - Biomarker for patient control - Google Patents

Abstract

An ex-vivo method to evaluate the efficacy of a treatment in a patient suffering from cancer with an immunogenic composition, where the test method comprises the step of: measuring interferon g levels in a blood, plasma or serum sample obtained from said patient after administration of said immunogenic composition to said patient, wherein the time between the start of said treatment comprising the administration of said immunogenic composition and measurements of interferon g is from about 4 to about 8 weeks; and where interferon g levels above about 4 pg / ml indicate that the patient is indicative of a satisfactory clinical outcome for treatment, and where said immunogenic composition contains at least one recombinant viral vector that expresses in whole all or part of the MUC1 antigen and where said recombinant viral vector also expresses IL-2.

Description

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DESCRIPTION

Biomarker for patient control

The present invention is included in the field of immunotherapy and refers to methods for determining the efficacy of certain immunotherapeutic treatments. The methods of the invention include the measurement of a special biomarker at a certain time, after starting the immunotherapeutic treatment, to evaluate the clinical outcome of said treatment. Therefore, the invention has applications in the medical field.

Traditional vaccination techniques have been known for many years that involve the introduction of an antigen (eg, peptides, proteins) into an animal system that can induce an immune response and thus protect! to said animal, for example, against infection. These techniques have been subsequently included in the development of vaccines, both live and inactivated. Live vaccines are typically non-pathogenic attenuated versions of an infectious agent, which are capable of initiating an immune response directed against a pathogenic version of the infectious agent.

In recent years there have been advances in the development of recombinant vaccines, especially living recombinant vaccines, in which foreign agents of interest are encoded and expressed from a vector. Among them, recombinant virus-based vectors have proven very promising and play an important role in the development of new vaccines. The ability to express proteins from foreign pathogens or tumor tissue, and to induce specific responses in vivo against these agents has been investigated in many viruses. Generally, these gene vaccines can stimulate potent humoral and cellular immune responses and viral vectors could be an effective strategy, both for the release of genes encoding antigens, and for the facilitation and improvement of antigen presentation. To be used as a vaccine vehicle, the ideal viral vector must be safe and allow an effective presentation of the specific pathogen antigens required to the immune system. In addition, the vector system must meet criteria that allow large-scale production. To date, several viral vaccine vectors have emerged, all of them with relative advantages and limits, depending on the proposed application (for a review on recombinant viral vaccines, see, for example, Harrop and Carroll, 2006, Front Biosci ., 11, 804-817; Yokoyama et al., 1997, J Vet Med Sci., 59, 311-322). The use of such recombinant vaccines is commonly called targeted immunotherapy or specific antigen-specific active immunotherapy.

Following the observation, in the early 1990s, that plasmid DNA vectors could directly transfect animal cells in vivo, significant efforts have also been made in research to develop immunotherapeutic techniques based on the use of DNA plasmids to induce an immune response, by direct introduction into DNA animals encoding antigens. Such techniques, which are widely mentioned as DNA vaccination or DNA immunotherapy, have now been used to elicit immunoprotective responses in a large number of disease models. For a review on DNA vaccines, see Reyes-Sandoval and Ertl, 2001 (Current Molecular Medicine, 1, 217-243).

However, in the field of immunotherapy a general problem has been identified on a means to induce in the treated individuals an immune response strong enough to protect them against infections and diseases.

Therefore, in recent years there has been an important effort to discover new pharmacological compounds that act by stimulating certain key aspects of the immune system that will serve to increase the immune response induced by immunotherapies. Most of these compounds, which are called adjuvants or immune response modifiers (MRIs), appear to act through basic mechanisms of the immune system, using Toll-like receptors (TLRs, Toll-like receptors) to induce biosynthesis of various important cytokines (e.g., interferons, interleukins, tumor necrosis factor, etc. See, for example, Schiller et al., 2006, Exp Dermatol., 15, 331-341). It has been shown that these compounds stimulate a rapid release of certain cytokines derived from dendritic cells, monocytes and macrophages and that they are also able to stimulate B cells to secrete antibodies that play an important role in the antiviral and antitumor activities of MRI compounds .

As an alternative, immunotherapeutic strategies have been proposed, most of them based on a primary stimulation vaccination regimen. According to these "primary stimulus" immunotherapy protocols, the immune system is first induced by administering a sensitizing composition to the patient and then reinforced by administering a second reinforcing composition (see, for example, EP1411974 or US20030191076)

In addition, in the healthcare context, it has been shown that a treatment can be effective only in a specific group of patients.

For example, WO 2007/015171 describes the treatment of non-microclinical lung cancer with a liposomal formulation comprising MUC1 and IL-2 and communicates that, if after treatment with said

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composition, one patient had a positive proliferative response of MUC1 specific T cells, the average survival time of these responding patients increased significantly compared to those who did not respond.

It is therefore desirable to provide doctors with tools and methods that allow them to adapt personalized optimal therapies for the patient, that is, prescribe the appropriate therapy to the appropriate patient at the right time, to provide a greater success rate of the treatment, control the response to treatment, increase the efficacy and pharmacological safety, eliminate unnecessary treatment of patients for whom the therapy is not appropriate, avoid unnecessary side effects and toxicity to the patient, reduce the cost of an ineffective medication for patients and insurers , unnecessary or dangerous, and improve the patient's quality of life, causing cancer to end up being a controlled disease, with appropriate follow-up trials.

In this regard, the bibliography proposes various tools and methods, such as, for example:

- Pharmacogenetics, which consists in the study of the individual response to drugs based on genetic differences. These responses refer to how a drug works in any given individual, how it is metabolized, its toxicity and dosage requirements. With the human genome project, pharmacogenetics has expanded to pharmacogenomics. Pharmacogenomics goes beyond pharmacogenetics, with the possibility of finding uses from the discovery and development of drugs, the discovery and validation of targets, and clinical trials;

- Metabolomics can also be applied to the field of predictive medicine. Unlike pharmacogenetics, which is limited to genetic factors, pharmacometabolomics can predict an individual's response to a drug, based not only on genetic factors, but also on non-genetic factors, such as other drugs in the patient's organism. , the patient's current state of health, etc.

- The role of biomarkers is becoming increasingly important in the clinical development of treatments. A biomarker can be an indicator of normal biological processes, disease processes, or pharmacological responses to therapeutic intervention. Its role ranges from stratification of the patient population to help identify the patients who respond to those who do not respond until the determination of the effectiveness of the therapy. Biomarkers can be a valuable tool for making better decisions that will reduce the cost of drug development and allow therapies to reach the appropriate population of patients faster. The invention provides materials and methods as described hereinbelow to evaluate the efficacy of a treatment that involves the administration of an immunogenic composition to a patient (i.e., an immunotherapy treatment) using biological markers (biomarkers) that are has determined that they are a substantially reliable badge that correlates with the desired immune response.

Therefore, the present invention relates to

an ex-vivo method to evaluate the efficacy of a treatment in a patient suffering from a cancer with an immunogenic composition, comprising the steps of:

measuring interferon g levels in a blood, plasma or serum sample, obtained from said patient after the administration of said immunogenic composition to said patient, where the time between the start of said treatment comprising the administration of said immunogenic composition and Interferon g measurements are approximately 4 to approximately 8 weeks; Y

where interferon g levels above 4 pg / ml indicate that the patient is representative of a satisfactory clinical result for treatment, and where said immunogenic composition contains at least one recombinant viral vector that expresses in vivo all or part of the antigen of MUC1, and where said recombinant viral vector also expresses IL-2.

The ability to predict the medical outcome of a treatment, as described above, shortly after its onset, will allow medical professionals and patients to identify an ineffective therapy, make informed decisions regarding the course of treatment, such as if abandons or is allowed to apply an alternative therapy.

As used herein throughout the entire application, the terms "un", "one" and "one" are used to mean "at least one (a)", "at least one ( a) first (a), “one (a) or more” or “a plurality” of the referred compounds or stages, unless the context dictates otherwise. For example, the expression "a cell" includes a plurality of cells that includes a mixture thereof. More specifically, "at least one (a)" and "one (a) or more," means a number that is one or more than one, with a special preference for one, two or three.

The expression "and / or", wherever used herein, includes the meaning of "and", "or" and "any other combination of the elements related to said expression."

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The terms "about" or "about", in the manner in which they are used herein, mean within 20%, preferably within 10%, and more preferably within 5%.

The terms "patient" and "subject" refer to a vertebrate, particularly a member of the mammal species and includes, but are not limited to, pets, sports animals, primates, including humans.

In the manner in which they are used herein, the terms "treatment" or "treat" refer to therapy.

An "effective amount" or a "sufficient amount" of an active compound is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations. A "therapeutically effective amount" is an amount to effect beneficial clinical results.

In accordance with the invention, the term "evaluate" should be understood as "controlling, modifying or adjusting" a treatment that involves the administration of an immunogenic composition to a patient as defined hereinbefore.

In certain aspects, the method may also include measuring the interferon levels of a patient before immunotherapeutic treatment.

The time between the start of immunotherapeutic treatment and interferon g measurements is approximately 4 weeks to approximately 8 weeks. In a preferred embodiment of the invention, the time interval is approximately 5 weeks. Likewise, additional measurements (ie, a third, a fourth, a fifth, etc. measurement) can be made at similar time intervals after the second measurement.

According to a special embodiment of the invention, the "immunotherapeutic treatment" consists of at least one administration of said immunogenic composition to said patient. According to a special embodiment, the "immunotherapeutic treatment" consists of successive administrations of said immunogenic composition to said patient, more specifically, it consists of the weekly administration for at least 2 weeks, preferably for at least 6 weeks.

The method includes determining the levels of interferon g in a patient after the administration of said immunogenic composition thereto, comparing said levels with a threshold value and evaluating the efficacy of the immunotherapeutic treatment based on the levels of interferon g compared with the threshold value

The threshold value and / or the detection limit is approximately 4 pg / ml (eg, 4.6 pg / ml in plasma), preferably measured by multi-analyte profiling of plasma proteins, using the Luminex® system (see Example 1). The expert who uses another test / method (eg, ELISA), will not have difficulty in determining the equivalent for a specific test of said threshold value and / or detection limit of approximately 4 pg / ml measured by multi-analyte prothelial profiling. plasma, using the Luminex® system. According to special embodiments, said threshold value according to the invention can be defined as a threshold value equivalent to the threshold value measured by multi-analyte profiling of plasma proteins, using the Luminex® system. By a "threshold value" equivalent to the threshold value measured by multi-analyte profiling of plasma proteins, using the Luminex® system ", a threshold value is identified that identifies the same patients as those identified by the threshold value measured by multi-analyte profiling of plasma prothelins. , using the Luminex® system.

According to a special embodiment, the invention relates to a method described hereinbefore, wherein said treatment with said immunogenic composition comprises the administration of one or more doses of said immunogenic composition to said subject.

In accordance with an alternative embodiment of the invention, the method of the invention also comprises an initial step which consists in determining the levels of interferon g in the patient's organism before the administration of said immunogenic composition.

In accordance with the present invention, the level of interferon g is measured in a sample of blood, plasma or serum obtained from the patient.

The methods of obtaining blood or serum are well known in the art and are not part of the invention.

In addition, numerous methods for detecting and quantifying polypeptides, in particular interferon g, are known. Such methods are, but are not limited to, antibody-based methods, more specifically, methods based on monoclonal antibodies. Particular methods for detecting and quantifying interferon g are not important for the invention. For example, the materials and methods of the present invention can be used with the technology.

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Luminex (Luminex Corporation, Austin, Tex.), Or adsorption enzyme immunoassay (ELISA), numerous ELISA kits are available on the market, eg, from CliniScience, Diaclone, Biosource).

As used herein, the terms "immunogenic composition", "vaccine composition", "vaccine", or similar expressions can be used interchangeably.

Said immunogenic composition contains at least one recombinant viral vector that expresses in vivo all or part of the MUC1 antigen and where said recombinant viral vector expresses, in addition IL-2.

According to another embodiment, the immunogenic composition also comprises at least one modifier of the immune response. Examples of such immune response modifiers (MRIs) are CpG oligonucleotides (see, for example, US 6,194,388; US2006094683; WO 2004039829), lipopolysaccharides, polyinosic acid complexes: polycyclic acid (Kadowaki, et al., 2001 J. Immunol. 166, 2291-2295), and polypeptides and proteins that are known to induce the production of cytokines from dendritic cells and / or monocytes / macrophages. Other examples of such immune response modifiers (MRIs) are small organic molecules, such as imidazoquinolinamines, imidazopyridine amines, cycloalkylimidazopyridine amines condensed in 6, 7, imidazonaphthyridine amines, oxazoloquinoline amines, thiazoloquinoline aminoquinoline amines and amines bridge 1,2 (see, for example, US 4,689,338; US 5,389,640; US 6,110,929; and US 6,331,539).

If necessary, the nucleic acid molecule of use in the invention can be optimized to provide high levels of MUC1 antigen expression in a particular host cell or organism, e.g. eg, a human host cell or organism. Typically, codon optimization is performed by replacing one or more corresponding "native" codons with a codon of infrequent use in the mammalian host cell with one or more codons encoding the same amino acid, the use of which is more frequent. This can be achieved by conventional mutagenesis or by chemical synthesis techniques (eg producing a synthetic nucleic acid). It is not necessary to replace all the corresponding native codons with the codons of infrequent use, since an increased expression can be achieved even with a partial replacement. In addition, some deviations from strict adherence to optimized codon usage can be made to accommodate the introduction of one or more restriction sites.

As used herein, the term "recombinant vector" refers to viral vectors.

Particularly important in the context of the invention are vectors for use in genetic therapy (ie, that are capable of releasing nucleic acid in a host organism), as well! as the expression vectors for use in various expression systems.

Appropriate viral vectors may come from a variety of different viruses (eg, retroviruses, adenoviruses, VAA, poxviruses, herpesviruses, measles virus, spongiform viruses and the like). As used herein, the expression "viral vector" encompasses DNA / RNA vectors, as! as viral particles generated by them. Viral vectors may be competent for replication or be genetically disabled so that their replication is altered or they have no replication capability. The term "competent for replication," as used herein, encompasses selective and conditionally replicative replication vectors, which have been designed to replicate better or selectively in specific host cells (eg, tumor cells).

In one aspect, the recombinant vector for use in the invention is a recombinant adenoviral vector (for a review, see "Adenoviral vectors for gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press). It can come from a variety of human or animal sources and any serotype of adenovirus serotypes 1 to 51 can be employed. Particularly, human adenoviruses 2 (Ad2), 5 (Ad5), 6 (Ad6), 11 (Ad11) are preferred , 24 (Ad24) and 35 (Ad35). These adenoviruses can be obtained from the American Type Culture Collection (ATCC, Rockville, Md.), And have been the subject of numerous publications describing their sequence, organization and production methods, allowing the expert to apply them ( see, for example, documents US 6,133,028; US 6,110,735; WO 02/40665; WO 00/50573; EP 1016711; Vogels et al., 2003, J. Virol. 77, 8263-8271).

The adenoviral vector for use in the present invention may be competent for replication. Those skilled in the art can readily have numerous examples of adenoviral vectors competent for replication (see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727). They can be designed, for example, from a natural adenovirus genome, by deletion in the E1A CR2 domain (see, for example, WO00 / 24408) and / or by replacing the native E1 and / or E4 promoters with tissue promoters, Tumor or cell state specifics (see, for example, US 5,998,205, WO99 / 25860, US 5,698,443, WO00 / 46355, WO00 / 15820 and WO01 / 36650).

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Alternatively, the adenoviral vector for use in the invention has defective replication capability (see, for example, WO94 / 28152; Lusky et al., 1998, J. Virol 72, 2022-2032). Preferred adenoviral vectors with defective replication capability are defective for E1 (see, for example, US 6,136,594 and US 6,013,638), with an E1 deletion ranging from approximately positions 459 to 3328, or approximately from positions 459 to 3510 (by reference to the sequence of human adenovirus type 5 disclosed in GeneBank with the registration number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280285). The cloning capacity can be further improved by deleting one or more additional portions of the adenoviral genome (all or part of the non-essential region E3 and other essential regions E2, E4). An insertion of a nucleic acid can be made at any location of the adenoviral vector by homologous recombination, as described in Chartier et al. (1996, J. Virol. 70, 4805-4810). For example, the nucleic acid encoding the HPV-16 E6 polypeptide may be inserted in place of the E1 region, and the nucleic acid encoding the HPV-16 E7 polypeptide in place of the E3 region, or vice versa.

In another preferred aspect, the use vector in the invention is a poxviral vector (see, for example, Cox et al. In "Viruses in Human Gene Therapy" Ed J. M. Hos, Carolina Academic Press). According to another preferred embodiment, this is selected from the group consisting of the vaccine virus; Suitable vaccine viruses include, without limitation, the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), the strain Wyeth and the highly attenuated attenuated virus derived therefrom, including the MVA virus (Modified Vaccinia Ankara) (for a review, see Mayr, A., et al., 1975, Infection 3, 6-14) and derivatives thereof (such as vaccine MvA strains 575 (ECaCc V00120707 - US 6,913,752), NYVAC (see WO 92/15672-Tartaglia et al., 1992, Virology, 188, 5 217-232). The complete sequence of the genome of the MVA and the comparison with the genome of the Copenhagen W strain has allowed the exact identification of the seven deletions (I to VII) that occurred in the genome of the MVA (Antoine et al., 1998, Virology 244, 365-396), any of which can be used to insert the nucleic acid encoding the antigen.The vector can also be obtained from any other family member lia Poxviridae, in particular, of avian smallpox (p. eg, TROVAC, see Paoletti et al., 1995, Dev Biol Stand., 84, 159-163); canarypox (eg, ALVAC, WO 95/27780, Paoletti et al., 1995, Dev Biol Stand., 84, 159-163); pigeon pox; swine and similar smallpox. By way of example, those skilled in the art can refer to WO 92 15672 (incorporated by reference) which describes the production of expression vectors based on poxviruses capable of expressing said heterologous nucleotide sequences, especially the nucleotide sequences encoding the antigen.

The basic technique for inserting the nucleic acid and associated regulatory elements necessary for expression in a poxviral genome is described in numerous documents accessible to those skilled in the art (Paul et al., 2002, Cancer gene Ther. 9, 470-477 ; Piccini et al., 1987, Methods of Enzymology 153, 545-563; US 4,769,330; US 4,772,848; US 4,603,112; US 5,100,587 and US 5,179,993). Usually, it is carried out through homologous recombination between overlapping sequences (i.e., the desired insertion site) present both in the viral genome and in a plasmid that carries the nucleic acid to be inserted.

The nucleic acid encoding the MUC1 antigen is preferably inserted into a non-essential locus of the poxviral genome, so that the recombinant poxvirus remains viable and infectious. Non-essential regions are non-coding intergenic regions, or any gene in which inactivation or deletion does not significantly impair viral growth, replication or infection. The insertion in an essential viral locus can also be contemplated, since the defective function is facilitated in trans during the production of viral particles, for example, using a collaborative cell line that carries the complementary sequences corresponding to those deleted in the poxviral genome.

When the Copenhagen strain of the vaccine virus is used, the nucleic acid encoding the antigen is preferably inserted into the thymidine kinase (tc) gene (Hruby et al., 1983, Proc. Natl. Acad. Sci USA 80, 3411- 3415; Weir et al., 1983, J. Virol. 46, 530-537). However, other insertion sites are also appropriate, e.g. eg, in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189-4198), in the K1L locus, in the u gene (Zhou et al., 1990, J. Gen. Virol .71, 2185-2190) or at the far left of the genome of the vaccine virus, where various spontaneous or designed deletions have been published in the bibliography (Altenburger et al., 1989, Archives Virol. 105, 15-27; Moss et al. . 1981, J. Virol. 40, 387-395; Panicali et al., 1981, J. Virol. 37, 1000-1010; Perkus et al., 1989, J. Virol. 63, 3829-3836; Perkus et al. ., 1990, Virol. 179, 276-286; Perkus et al., 1991, Virol. 180, 406-410).

When MVA is used, the nucleic acid encoding the antigen can be inserted into any one of the deletions I to VII identified, as! as in locus D4R, but insertion in deletion II or III is preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038; Sutter et al., 1994, Vaccine 12, 1032-1040 )

When avian smallpox virus is used, although insertion into the thymidine kinase gene can be considered, the nucleic acid encoding the antigen is preferably introduced into the intergenic region between the ORFs (Open Reading Frame, open reading phase) 7 and 9 (see, for example, documents EP 314 569 and US 5,180,675).

According to a special embodiment, said recombinant viral vector is a recombinant adenoviral vector.

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According to another special embodiment, said recombinant viral vector is a recombinant vaccine vector

According to a preferred embodiment, said recombinant viral vector is a recombinant MVA vector

Preferably, the nucleic acid encoding the MUC1 antigen used in the invention is in a form suitable for expression in a host cell or organism, which means that the nucleic acid sequence encoding the MUC1 antigen is placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism. As used herein, the term "regulatory sequence" refers to any sequence that allows, contributes or modulates the expression of a nucleic acid in a particular host cell, including replication, duplication, transcription, cutting and splicing, translation, stability and / or transport of the nucleic acid or one of its derivatives (that is, mRNA) within the host cell. Those skilled in the art will appreciate that the choice of regulatory sequences may depend on factors such as the host cell, the vector and the level of expression desired. The nucleic acid encoding the MUC1 antigen is operatively linked to a gene expression sequence that directs the expression of the nucleic acid of the MUC1 antigen within a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence, or a promoter enhancer combination that facilitates the effective transcription and translation of the nucleic acid of the MUC1 antigen to which it is operably linked. The gene expression sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Mamlfer constitutive promoters include, but are not limited to, promoters of the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters. An example of viral promoters that function constitutively in eukaryotic cells are, for example, cytomegalovirus (CMV) promoters, simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Those skilled in the art know other constitutive promoters.

Promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothioneelna promoter is induced to promote transcription and translation in the presence of certain metal ions. Those of ordinary skill in the art know other inducible promoters. In general, the gene expression sequence will necessarily include 5 'non-translatable and 5' non-translatable sequences involved in the initiation of transcription and translation respectively, such as a TATA box, a terminal protective sequence, a CAAT sequence, and Similar. Especially, said 5 'non-translatable sequences will include a promoter region that includes a promoter sequence for the transcriptional control of the nucleic acid of the operably linked antigen. The gene expression sequences will optionally include enhancer sequences or upstream activator sequences as appropriate. Preferred promoters for use in a poxviral vector (see below) include, without limitation, vaccine promoters 7.5 K, H5R, TK, p28, p11 and K1L, chimeric promoters between early and late poxviral promoters, as! as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and in Kumar and Boyle (1990, Virology 179, 151-158).

The promoter is of special importance and the present invention encompasses the use of constitutive promoters that direct the expression of nucleic acid in many types of host cells and those that direct the expression only in certain host cells or in response to specific exogenous events or factors ( e.g. temperature, addition of nutrients, hormones or other ligand). Suitable promoters are broadly described in the literature and viral promoters such as RSV, SV40, CMV and MLP promoters can be cited more specifically. Preferred promoters for use in a poxviral vector include, without limitation, 7.5 K, H5R, TK, p28, p11 and K1L vaccine promoters, chimeric promoters between early and late poxviral promoters, as well! as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158).

Those skilled in the art will appreciate that the regulatory elements that control the expression of the nucleic acid molecule encoding the MUC1 antigen may further comprise additional elements for a start, a regulation and / or a termination of transcription (e.g. ., poly-A transcription termination sequences) mRNA transport (e.g., nuclear localization signal sequences) processing (e.g., splice signals) and stability (e.g., introns , 5 'and 3' non-coding sequences) translation (eg, signal peptide, propeptide, tripartite llder sequences, ribosome binding sites, Shine-Dalgamo sequences, etc.,), in the host cell or organism.

The viral recombinant vector for use in the present invention further comprises a nucleic acid encoding IL-2. Such vector may comprise other nucleic acids encoding other cytokines. Suitable cytokines include, without limitation, interleukins (e.g., IL-7, IL-15, IL-18, IL-21) and interferons (e.g., IFNg, IFNa). Said nucleic acids expressing other cytokines can be transported through the recombinant viral vector encoding MUC1 and IL-2 or through an independent recombinant vector that can be of the same or of different origin.

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Infectious viral particles comprising the recombinant viral vector described above can be produced by a usual process. An example of a process includes the following stages:

to. introduction of the viral vector into a suitable cell line,

b. culture of said cell line under suitable conditions to allow the production of said infectious viral particle,

C. recovery of said cell line culture from the infectious viral particle produced, and

d. optionally, purification of said recovered infectious viral particle.

They are suitable cells for propagating adenoviral vectors, for example, 293 cells, PERC6 cells, HER96 cells, or cells such as those disclosed in WO 94/28152, WO 97/00326, US 6,127,175.

Appropriate cells for propagating poxviral vectors are bird cells and, more preferably, primary chicken embryo fibroblasts (FEP) prepared from chicken embryos obtained from fertilized eggs.

Infectious viral particles can be recovered from the culture supernatant or cells after lysis (e.g., by chemical means, freezing / thawing, osmotic shock, mechanical shock, ultrasound and the like). The viral particles can be isolated by consecutive rounds of purification on plates and then, they can be purified using the methods of the technique (chromatographic methods, gradient ultracentrifugation of cesium chloride or sucrose).

According to another embodiment, the methods of the invention can be combined with other methods to predict the efficacy of treatments, more specifically, to predict the efficacy of immunotherapeutic treatments. For example, biomarker levels, such as activated NK cell levels (see patent application claiming priority of document EP 08305876.8) or sICAM-1 levels (see patent application claiming priority of document EP 09305032.6 ).

If desired, administration of the immunogenic composition can be carried out together with one or more conventional therapeutic modalities (eg, radiation, chemotherapy and / or surgery). The use of multiple therapeutic approaches provides the selected patient with an intervention with a broader base. In one embodiment, the administration of the immunogenic composition may be preceded or followed by surgical intervention. In another embodiment, it may be preceded or followed by radiation therapy (eg, gamma radiation). Those skilled in the art can rapidly formulate appropriate radiotherapy protocols and parameters that can be used (see, for example, Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications that will be readily apparent to those skilled in the art). In yet another embodiment, the administration of the immunogenic composition is associated with chemotherapy with one or more drugs (e.g., drugs that are conventionally used to treat or prevent viral infections, pathological conditions related to viruses, cancer and the like).

Therefore, a method for improving the treatment of a cancer patient who is undergoing chemotherapy treatment with a chemotherapeutic agent is also disclosed, said method comprising the following steps:

- administration to the patient with cancer of one or more doses of an immunogenic composition, as described hereinbefore, and of one or more doses of a chemotherapeutic agent.

- measuring a level of interferon g in a blood, plasma or serum sample of said patient after at least one of said administrations of said immunogenic composition as described hereinbefore;

- where previous interferon g levels of approximately 4 pg / ml (eg 4.6 pg / ml) indicate that the subject is indicative of a satisfactory clinical result for treatment, that is, an increase in the Index of survival.

The administration of said chemotherapeutic agent can be done before the administration of said immunogenic composition, after the administration of said immunogenic composition, or simultaneously with the administration of said immunogenic composition.

In another embodiment, in the method of the invention, treatment with the immunogenic composition that the patient with cancer receives is carried out in accordance with a therapeutic modality of sensitization reinforcement comprising the sequential administration of one or more sensitizing compositions and one or more reinforcing compositions. Typically, sensitizing and reinforcing compositions use different vehicles that comprise or encode at least one common antigen domain. The sensitizing composition is initially administered to the host organism and the reinforcing composition is subsequently administered to the same host organism after a period ranging from one day to twelve months. Said sensitization reinforcement modality may comprise one to ten sequential administrations of the sensitizing composition.

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followed by one to ten sequential administrations of the reinforcement composition. If possible, injection intervals are a matter of one week to six months. In addition, the sensitizing and reinforcing compositions can be administered at the same site or at alternative sites through the same route of administration or through different routes.

According to a special realization, said cancer is, for example, breast cancer, colon cancer, kidney cancer, rectal cancer, lung cancer, head and neck cancer, renal cancer, malignant melanoma, larynx cancer, cancer ovarian cancer, cervical cancer, prostate cancer, non-microclinical lung cancer (NSCLC), hematological cancers, gastric cancers, myeloma.

According to a preferred embodiment, said cancer is non-microclinical lung cancer (NSCLC).

According to a special embodiment, an immune response directed towards the MUC1 antigen can be observed in the patient with treated cancer. According to one embodiment, said "immune response" in said patient population is directed towards different antigens.

According to another special embodiment, said "immune response" in said cancer patient is an immune response of T cells and, preferably, an immune response of CD8 + lymphocytes (cytotoxic T lymphocytes). According to another special embodiment, said "immune response" in said patient is an unspecific immune response. According to another special embodiment, said "immune response" in said patient is a stimulation of the innate immune response.

The ability to induce or stimulate an immune response after administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays that are customary in the art. For a general description of available techniques to evaluate the appearance and activation of an immune response, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed J Wiley & Sons Inc, National Institute of Health). Measurement of cellular immunity can be performed by measuring the profiles of cytokines secreted by activated effector cells, including those derived from CD4 + and CD8 + T cells (e.g., quantification of IL-10 or IFN gamma producing cells by ELIspot), determining the activation status of effector immune cells (eg, T cell proliferation assays by a classical thymidine captation [3H]), by testing antigen-specific T lymphocytes in a sensitized subject (eg, lysis specific for peptides in a cytotoxicity assay) or by detection of antigen-specific T cells by fluorescent MHC and / or multimer peptides (eg, tetramers). The ability to stimulate a humoral response can be determined by binding to antibodies and / or competition by binding to them (see, for example, Harlow, 1989, Antibodies, Cold Spring Harbor Press). The method of the invention can also be validated later in animal models exposed to an appropriate tumor inducing agent (eg, RMA cells expressing MUC1) to determine antitumor activity, which reflects an induction or potentiation of an anti-immune response. antigenic

Therefore, a method is also described for prolonging the survival of a patient with cancer treated by administering an immunogenic composition as defined hereinbefore, said method comprising the following steps:

- administration to a patient of one or more doses of an immunogenic composition, as defined hereinbefore.

- measurement of an interferon level of said patient after at least one of said administrations of said immunogenic composition, as described hereinbefore.

The use of interferon g as a biomarker is also disclosed to predict whether a cancer patient treated by administering an immunogenic composition, as described hereinbefore, is or is not susceptible to developing a therapeutic response, preferably an immune therapeutic response. as a consequence of said administration of an immunogenic composition.

In addition, the use of interferon g as a biomarker for controlling, modifying or adjusting a treatment involving the administration of an immunogenic composition, as defined hereinbefore, to a patient with cancer is disclosed.

The use of the interferon g level as a biomarker to control, modify or adjust a treatment involving the administration of an immunogenic composition as defined hereinbefore, to a patient with cancer, where the levels of an interferon g are also disclosed. , measured after such administration as defined herein, above 4 pg / ml (eg, 4.6 pg / ml), indicate that the patient is indicative of a satisfactory clinical result for treatment, that is, the increase in the survival rate.

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The present invention also relates to the use of a kit to analyze, in an ex-vivo method according to the present invention, if a patient suffering from a cancer is therapeutically responding to a method of treatment with an immunogenic composition, the kit comprising :

(a) antibodies to determine the level of interferon g in said blood, plasma or serum sample of the patient; Y

(b) instructions to interpret the data obtained by saying that interferon g levels above 4 pg / ml indicate that the subject is indicative of a satisfactory clinical outcome for treatment according to the method of the present invention;

wherein said immunogenic composition contains at least one recombinant viral vector that expresses in vivo all or part of the MUC1 antigen and where said recombinant viral vector also expresses IL-2; Y

wherein the time between the start of said treatment comprising the administration of said immunogenic composition and the measurements of interferon g is from about 4 to about 8 weeks.

Therefore, the invention also discloses kits that include parts to implement the methods described herein and that will be obvious from the examples provided herein. The kit (or kits) of parts, may include reagents to collect and measure serum interferon gamma levels. Such reagents are antibodies. The kits may also include instruments to collect and / or process biological samples. The kits also contain instructions for use, threshold values and / or instructions for their determination, and instructions for interpreting the data obtained from the use of the kits.

Examples

Figures:

Figure 1: Survival curves describing immunotherapy by vaccine in lung cancer: patients with or without interferon g detectable in day 43

- Group 1: Therapeutic vaccine (ie, immunogenic composition) + chemotherapy in patients with detectable interferon g at day 43. Detectable, defined as> Detection Limit, which is 4.6 pg / ml. 22 patients Median survival 25.4 months.

- Group 2: Therapeutic vaccine (ie, immunogenic composition) + chemotherapy in patients with interferon g not detectable at day 43. Not detectable, defined as <4.6 pg / ml sCD54 in peripheral blood. 29 patients Median survival 10.14 months.

Significant difference, by logarithmic range: p = 0.019 O Complete + Censored

Figure 2: Survival curves describing chemotherapy in lung cancer: patients with or without detectable interferon g (> 4.6 pg / ml)

- Group 1: Chemotherapy alone (without therapeutic vaccine) in patients with detectable interferon g at day 43. Detectable, defined as> 4.6 pg / ml in peripheral blood. 25 patients Median survival 8.5 months.

- Group 2: Chemotherapy alone (without therapeutic vaccine) in patients with interferon g not detectable at day 43. Not detectable, defined as <4.6 pg / ml of IFNg in peripheral blood. 34 patients Median survival 12.8 months

Significant difference, using logarithmic range: p = 0.047 O Complete + Censored

Figure 3: Survival curves describing chemotherapy in lung cancer: patients with> 4.6 pg / ml of interferon g

- Group 1: Therapeutic vaccine (ie, immunogenic composition) + chemotherapy in patients with detectable levels of interferon g. Detectable levels, defined as> 4.6 pg / ml in peripheral blood. 22 patients Median survival 25.4 months.

- Group 2: Chemotherapy alone (without therapeutic TG4010 vaccine) in patients with detectable levels of interferon g. Detectable levels, defined as> 4.6 pg / ml of interferon g in peripheral blood. 25 patients Median survival 8.5 months.

Significant difference, by logarithmic range: p = 0.002 O Complete + Censored

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The immunogenic composition, called the TG4010 vaccine, was used to treat patients with non-microclinical lung cancer (NSCLC) in combination with usual chemotherapy.

The TG4010 vaccine is a recombinant modified Ankara virus (MVA) that expresses both IL-2 and tumor-associated MUC1 antigen

One hundred and forty-eight patients were randomized to receive:

- chemotherapy (75 mg / m2 of cisplatin on day 1 and 1250 mg / m2 of gemcitabine on day 1 and day 8 every 3 weeks for up to 6 cycles), either alone (Study Group 2), or

- Chemotherapy together with TG4010 (Study Group 1)

Tumors were evaluated (WHO criteria) every 6 weeks. The assessment criteria were progression-free survival (SSP) at 6 months and overall survival with intention-to-treat analysis.

Blood samples were taken on day 43 (one week after the 6th weekly injection) and sent immediately to a central immunology laboratory where the plasma samples were divided into alliquots and frozen. The frozen plasma allcuotas were sent, in batches, on dry ice to a second central laboratory where interferon gamma levels were evaluated.

The content of interferon gamma (interferon g) of the plasma samples was evaluated by multi-analyte profiling of plasma proteins using the Luminex® system. The detection limit has been set as 4.6 pg / ml.

Figure 1 shows that patients [Group 1 (TG4010 + chemotherapy] with detectable interferon g on day 43 survive longer (median survival = 25.4 months) than patients with interferon g not detectable (median survival 10,14 months) when treated with both the TG4010 vaccine and chemotherapy.

The data in Figure 2 demonstrate that the positive association between detectable plasma interferon g and overall survival is limited to patients receiving the therapeutic vaccine. Patients in group 2, who only receive chemotherapy, who had detectable plasma interferon g on day 43, had worse survival (8.5 months) than patients with interferon g not detectable on day 43 (12.8 months).

The data in Figure 3 demonstrate that the effect of selecting patients based on the detection of interferon g in their plasma on day 43 is limited to patients receiving the therapeutic vaccine. Figure 3 shows that patients with detectable interferon g on day 43 have a higher survival expectation only if they receive the therapeutic vaccine.

Claims (14)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 1. An ex-vivo method to evaluate the efficacy of a treatment in a patient suffering from cancer with an immunogenic composition, where the test method comprises the stage of: measuring the levels of interferon g in a blood, plasma or serum sample obtained from said patient after the administration of said immunogenic composition to said patient, where the time between the start of said treatment comprising the administration of said immunogenic composition and the Interferon g measurements are from about 4 to about 8 weeks; Y where interferon g levels above about 4 pg / ml indicate that the patient is indicative of a satisfactory clinical result for treatment, and where said immunogenic composition contains at least one recombinant viral vector that expresses in whole all or part of the antigen MUC1 and where said recombinant viral vector also expresses IL-2. 2. The method of claim 1, wherein the level of interferon g is above about 4.6 pg / ml. 3. The method of claim 1 or 2, wherein the time between the start of said treatment comprising the administration of said immunogenic composition and measurements of interferon g is approximately 5 weeks. 4. The method of any one of claims 1 to 3, wherein said cancer is non-microclinical lung cancer (NSCLC). 5. The method of any one of claims 1 to 4, wherein said levels of interferon g are measured by multi-analyte profiling of plasma proteins using the Luminex® system. 6. The method of any one of claims 1 to 5, wherein said viral vector is competent for replication. 7. The method of any one of claims 1 to 5, wherein said viral vector has defective replication. 8. The method of claim 6 or 7, wherein said recombinant vector is a recombinant vaccine virus vector. 9. The method of claim 8, wherein said recombinant vaccine virus vector is a recombinant MVA vector. 10. The method of any one of claims 1 to 9, wherein said patient is a patient treated with a chemotherapeutic agent. 11. The method of claim 10, wherein the administration of said chemotherapeutic agent is done simultaneously with the administration of said immunogenic composition. 12. The method of any one of claims 1 to 11, wherein said method further comprises an initial step consisting in measuring the levels of interferon g in a blood, plasma or serum sample, obtained from said patient before of said administration of said immunogenic composition. 13. The use of a kit to analyze, in an ex-vivo method according to claim 1, if a patient suffering from cancer is therapeutically responding to a method of treatment with an immunogenic composition, wherein the kit comprises: (a) antibodies to determine the level of interferon g in said blood, plasma or serum sample of the patient; Y (b) instructions to interpret the data obtained by saying that the levels of interferon g above 4 pg / ml indicate that the subject is indicative of a satisfactory clinical result for treatment according to the method of claim 1; wherein said immunogenic composition contains at least one recombinant viral vector that expresses in vivo all or part of the MUC1 antigen, and wherein said recombinant viral vector also expresses IL-2; Y wherein the time between the start of said treatment, which comprises the administration of said immunogenic composition, and the measurements of interferon g, is from about 4 to about 8 weeks. 14. The use of claim 13, wherein the level of interferon g is above about 4.6 pg / ml.